User Guide
SPRIselect User Guide
SPRI Based Size Selection
B24965AA
October 2012
Beckman Coulter, Inc.
250 S. Kraemer Blvd.
Brea, CA 92821 U.S.A.
SPRIselect User Guide
PN B24965AA (October 2012)
© 2012 Beckman Coulter, Inc.
All rights reserved
The Beckman Coulter stylized logo, SPRI and SPRIselect
are trademarks of Beckman Coulter, Inc. and are
registered in the USPTO.
All other trademarks, service marks, products, or services
are trademarks or registered trademarks of their
respective holders.
Find us on the World Wide Web at:
www.beckmancoulter.com
Made in U.S.A.
Revision History
Initial Issue, 10/2012
SPRIselect User Guide version B24965AA
B24965AA
iii
Revision History
iv
B24965AA
Safety Notice
Do not attempt to perform any procedure before carefully reading all instructions. Always follow
product labeling and manufacturer’s recommendations. If in doubt as to how to proceed in any
situation, contact your Beckman Coulter Representative.
Alerts for Warning, Caution, Important, and Note
WARNING
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WARNING
WARNING indicates a potentially hazardous situation which, if not avoided, could
result in death or serious injury.
In this document the signal word WARNING is only used to indicate the possibility of personal
injury. It is not used to indicate the possibility of erroneous data.
CAUTION
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example the definition of CAUTION is in bold-face as shown below.
CAUTION
CAUTION indicates a potentially hazardous situation, which, if not avoided, may
result in minor or moderate injury. It may also be used to alert against unsafe
practices.
In this document the signal word CAUTION is used to indicate the possibility of damage to the
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IMPORTANT
The signal word IMPORTANT is in bold-face and the associated text (in this example the definItion
of IMPORTANT) is indented if it wraps.
IMPORTANT IMPORTANT is used for comments that add value to the step or procedure being performed.
Following the advice in the Important adds benefit to the performance of a piece of equipment or to a
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The signal word IMPORTANT is used to draw attention to information that is critical for the
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B24965AA
v
Safety Notice
Alerts for Warning, Caution, Important, and Note
NOTE
The signal word NOTE is in bold-face and associated text (in this example the definition of NOTE) is
indented if it wraps.
NOTE NOTE is used to call attention to notable information that should be followed during installation, use,
or servicing of this equipment.
vi
B24965AA
Contents
Revision History, iii
Safety Notice, v
Alerts for Warning, Caution, Important, and Note, v
WARNING, v
CAUTION, v
IMPORTANT, v
NOTE, vi
Introduction, ix
Introduction to the SPRIselect User Guide, ix
About This Manual, ix
Intended Use, ix
Warranty Disclaimer, ix
Conventions Used, ix
SPRI Based Size Selection, 1
Introduction, 1
Materials, 1
Sample Prerequisites, 2
Left Side Size Selection, 3
Right Side Size Selection, 5
Double Size Selection Considerations, 8
Effects of Common Laboratory Reagents on Size Selection, 10
Index
vii
Contents
viii
Introduction
Introduction to the SPRIselect User Guide
About This Manual
The information in this manual is organized as follows:
SPRI Based Size Selection
Provides an overview of SPRIselect, its uses and its key features.
Glossary
Provides definitions for terms used throughout this manual.
Intended Use
SPRIselect is intended for molecular biology research applications. It is not intended or validated
for use in the diagnosis of disease or other conditions.
Warranty Disclaimer
Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to
the method, including but not limited to warranties of fitness for a particular purpose or
merchantability or that the method is non-infringing. All other warranties are expressly
disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter.
Conventions Used
This manual uses the following conventions:
• The names of instrument manuals referred to in the text are in italic.
• Messages that appear on the instrument screen are in italic.
• Buttons that appear on the instrument screen are in bold face.
• Selections that appear on the instrument screen are in bold face.
• The software path to a specific function or screen appears with the greater than ( > ) symbol
between succeeding screen options, like this: Options > Log Configuration.
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ix
Introduction
Introduction to the SPRIselect User Guide
x
B24965AA
SPRI Based Size Selection
Introduction
SPRIselect is a SPRI-based chemistry that speeds and simplifies nucleic acid size selection for
fragment library preparation for Next Generation sequencing. In this process, size selection is
required to produce a uniform distribution of fragments around an average size. Using SPRIselect,
the size distribution can be adjusted to suit the application and platform used. The process can be
scaled for low to high throughput workflows.
The guidance provided below can be used to optimize the desired size selection range. Used
manually or automated on a liquid handling system such as the Biomek Laboratory Automation
Workstation, SPRIselect will provide rapid and consistent size selection suitable for most
applications.
SPRIselect is covered by US Patents 5705628, 6534262, and 5898071.
Materials
• 85% Ethanol, non-denatured (ethanol is hygroscopic, prepare fresh for optimal results)
• Water (molecular biology grade) or a standard buffer solution such as Tris (10 mM, pH 8) or TE
(10 mM Tris, pH 8, 1 mM EDTA) for DNA elution
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1-1
SPRI Based Size Selection
Sample Prerequisites
Sample Prerequisites
• Samples should be fragmented double-stranded DNA.
• Samples should be dissolved in molecular biology grade water or standard buffer solution such
as Tris or TE. See Effects of Common Laboratory Reagents on Size Selection at end of user guide for
more information.
• Sample volume should be ≥ 50 μL. A lower volume will decrease pipetting accuracy of
SPRIselect, therefore increasing selection point variability.
• DNA fragments may be size selected in a range no smaller than 150 bp and no larger than 800 bp.
• To maximize recovery for a Left Side Size Selection, the majority of the sample’s size
distribution should be larger than the selection point.
• To maximize recovery for a Right Side Size Selection, the majority of the sample’s size
distribution should be smaller than the selection point.
• To maximize recovery for a Double Size Selection, the size distribution should be centered
between the selection points.
1-2
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SPRI Based Size Selection
Left Side Size Selection
Left Side Size Selection
As a general rule, increasing the ratio of SPRIselect volume to sample volume will increase the
efficiency of binding smaller fragments. Figure 1 illustrates this relationship.
Figure 1 Agilent High Sensitivity DNA chip Electropherogram.
M = upper and lower markers for High Sensitivity DNA chip
Shear = 1 μL of 20 ng/μL input control sample in water
1.2x to 0.4x = 1 μL of shear, size selected with given ratio of SPRIselect volume to sample
volume.
Figure 2
1. Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads. Following the trend
depicted in Figure 1, add the required volume of SPRIselect for the desired ratio to the sample.
TIP Volume of sample * ratio = volume of SPRIselect.
Example: 50 μL sample * 0.8x ratio = 40 μL of SPRIselect
2. Mix the total reaction volume by pipetting 10 times and incubate at RT for 1 minute
OR
B24965AA
1-3
SPRI Based Size Selection
Left Side Size Selection
vortex for 1 minute at an appropriate speed until homogenous (depending on labware and total
volume).
NOTE Insufficient mixing of sample and SPRIselect will lead to inconsistent size selection results. Make
sure to mix well.
3. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to
settle to the magnet. Settle times will vary; a higher initial sample volume, higher SPRIselect
ratio or weaker magnets will require a longer settle time. Remove and discard the clear
supernatant.
NOTE Care should be taken not to aspirate more than a trace amount of beads during this step, as the
desired library is associated with the beads. Significant bead loss will result in reduced yield.
4. With the reaction vessel still on the magnet, add 180 μL of 85% ethanol (non-denatured) and
incubate at RT for 30 seconds. Remove and discard the ethanol supernatant.
NOTE Care should be taken not to aspirate more than a trace amount of beads during this step, as the
desired library is associated with the beads. Significant bead loss will result in reduced yield.
5. To elute the sample:
a.
Remove the reaction vessel from the magnet and add ≥ 20 μL of molecular biology grade
water or standard buffer solution such as Tris or TE.
NOTE Elution volume should be large enough so that the liquid level is high enough for the beads
to settle to the magnet.
b. Mix the total elution volume by pipetting 10 times to resuspend the beads and incubate at
RT for 1 minute
OR
vortex for 1 minute at an appropriate speed until homogenous (depending on labware and
total volume).
c. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI
beads to settle to the magnet. Settle times will vary; a higher elution volume or weaker
magnets will require a longer settle time.
6. Transfer the eluate (size selected sample) to an appropriate storage vessel.
1-4
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SPRI Based Size Selection
Right Side Size Selection
Right Side Size Selection
As a general rule, increasing the ratio of SPRIselect volume to sample volume will decrease the
efficiency of binding larger fragments. Figure 3 illustrates this relationship.
Figure 3 Agilent High Sensitivity DNA chip Electropherogram.
M = upper and lower markers for High Sensitivity DNA chip.
Shear = 1 μL of 20 ng/μL input control sample in water.
1.2x to 0.4x = 1 μL of shear, size selected with given ratio of SPRIselect volume to sample
volume.
Figure 4
1. Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads. Following the trend
depicted in Figure 3, add the required volume of SPRIselect for the desired ratio to the sample.
TIP Volume of sample * ratio = volume of SPRIselect.
Example: 50 μL * 0.7x ratio = 35 μL of SPRIselect
2. Mix the total reaction volume by pipetting 10 times and incubate at RT for 1 minute
OR
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1-5
SPRI Based Size Selection
Right Side Size Selection
Vortex for 1 minute at an appropriate speed until homogenous (depending on labware and total
volume).
NOTE Insufficient mixing of sample and SPRIselect will lead to inconsistent size selection results. Make
sure to mix well.
3. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI beads to
settle to the magnet. Settle times will vary; a higher initial sample volume, higher SPRIselect
ratio or weaker magnets will require a longer settle time.
4. Transfer the clear supernatant, which contains the Right Side Size Selected sample, to a new
reaction vessel. The reaction vessel with the remaining beads can be discarded.
NOTE Care should be taken not to aspirate more than a trace amount of beads during this step as the
undesired larger fragment sizes are associated with the beads. Significant bead transfer will cause
tailing into the larger size range.
5. Add the required volume of SPRIselect, using the calculation below, to the supernatant from
Step 4 above. This will bind the fragments in the supernatant to the new SPRI beads.
TIP Sample Volume μL * (1.8x – the initial ratio) = volume of SPRIselect
Example: 50 μL * (1.8 – 0.7) = 55 μL of SPRIselect
6. Perform the following:
a. Mix the total reaction volume by pipetting 10 times and incubate at RT for 1 minute
OR
vortex for 1 minute at an appropriate speed until homogenous (depending on labware and
total volume).
NOTE Insufficient mixing of sample and SPRIselect will lead to inconsistent size selection results.
b. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI
beads to settle to the magnet. Settle times will vary; a higher initial sample volume, higher
SPRIselect ratio or weaker magnets will require a longer settle time.
c. Remove and discard the clear supernatant.
NOTE Care should be taken not to aspirate more than a trace amount of beads during this step, as the
desired library is associated with the beads. Significant bead loss will result in reduced yield.
7. With the reaction vessel still on the magnet, add 180 μL of 85% ethanol (non-denatured) and
incubate at RT for 30 seconds. Remove and discard the ethanol supernatant.
NOTE Care should be taken not to aspirate more than a trace amount of beads during this step, as the
desired library is associated with the beads. Significant bead loss will result in reduced yield.
8. To elute the sample:
a. Remove the reaction vessel from the magnet and add ≥ 20 μL of molecular biology grade
water or standard buffer solution such as Tris or TE.
NOTE Elution volume should be large enough so that the liquid level is high enough for the beads
to settle to the magnet.
1-6
B24965AA
SPRI Based Size Selection
Right Side Size Selection
b. Mix the total elution volume by pipetting 10 times to resuspend the beads and incubate at
RT for 1 minute
OR
vortex for 1 minute at an appropriate speed until homogenous (depending on labware and
total volume).
c. Place the reaction vessel on an appropriate magnetic stand or plate and allow the SPRI
beads to settle to the magnet. Settle times will vary; a higher elution volume or weaker
magnets will require a longer settle time.
9. Transfer the eluate (size selected sample) to an appropriate storage vessel.
B24965AA
1-7
SPRI Based Size Selection
Double Size Selection Considerations
Double Size Selection Considerations
As a general rule, the Left Side Size Selection ratio always needs to be greater than the Right Side
Size Selection ratio. Figure 5 illustrates this relationship.
Figure 5 Agilent High Sensitivity DNA chip Electropherogram.
M = upper and lower markers for High Sensitivity DNA chip
Shear = 1 μL of 20 ng/μL input control sample in water
0.XX to 0.XX = 1 μL of shear, size selected with given Left side ratio and Right side ratio of
SPRIselect volume to sample volume
Table 1 Typical Recovery (determined by experiment)
Percentages calculated from Agilent 2100 Expert Smear Region analysis Conc. [pg/μL]
Table 1 Definitions:
Ratios (Left-Right): Left Side Size Selection ratio - Right Side Size Selection ratio
bp Region: Agilent 2100 Expert Smear Region analyzed for the given Ratios (Left-Right)
Selection Delta (bp): The difference between the Left and Right selection points of the bp Region
Example: 660 Right bp – 230 Left bp = 430 bp Delta
1-8
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SPRI Based Size Selection
Double Size Selection Considerations
Table 2 Recovery of Shear- Note: the 0.8-0.61 ratio sample seen in Table 1 will be used for all definition
examples below
Definition
B24965AA
bp Region’s
% of Shear
The
maximum
potential
recovery of
the Stock
sample for
the targeted
size selection
region.
Recovered %
of bp Region
Percent
recovery of
the targeted
region
Recovered %
of Shear
Percent
recovery of
the Shear
sample.
Diagram
1-9
SPRI Based Size Selection
Effects of Common Laboratory Reagents on Size Selection
Effects of Common Laboratory Reagents on Size Selection
The common laboratory reagents described in Table 3 were added to the reaction (as an additive)
to determine the effect on Size Selection.
•
Each Left Side Size Selection below was 1 μg sheared in 50 μL + 50 μL additive + 65 μL SPRIselect
(0.65x ratio). Final elution in 100 μL of water. 1 μL of eluent was run on an Agilent High
Sensitivity DNA chip.
TIP If a possible reagent additive not listed below is of concern, use this dilution scheme to test if there
is any effect on size selection.
• The “0.65x Control” (red trace) is the expected selection result for a 0.65x Left Side Size
Selection.
• The concentration of the additive can be found in the Electropherogram table.
• The “M” is the Agilent High Sensitivity DNA assay’s upper and lower markers.
• Keep in mind that additive effects are cumulative. If there are multiple additives that all move
the selection the same direction, the result will be larger than any individual component. If
some component(s) move the selection Left and some Right, it’s possible to have little to no
effect.
1-10
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SPRI Based Size Selection
Effects of Common Laboratory Reagents on Size Selection
Table 3 Effects of Common Laboratory Reagents on Size Selection. Blue background represents wider
selection. Red background represents narrower selection.
B24965AA
Additive
Effect
DMSO
(Dimethyl
sulfoxide)
None
DTT
(Dithiothreitol)
None
85% Ethanol
Wider Size
Selection
Agilent High Sensitivity Chip Trace
1-11
SPRI Based Size Selection
Effects of Common Laboratory Reagents on Size Selection
Table 3 Effects of Common Laboratory Reagents on Size Selection. Blue background represents wider
selection. Red background represents narrower selection.
1-12
Additive
Effect
Glycerol
Narrower Size
Selection
KCI
(Potassium
chloride)
None
MgCl2
(Magnesium
chloride)
Wider Size
Selection
Agilent High Sensitivity Chip Trace
B24965AA
SPRI Based Size Selection
Effects of Common Laboratory Reagents on Size Selection
Table 3 Effects of Common Laboratory Reagents on Size Selection. Blue background represents wider
selection. Red background represents narrower selection.
B24965AA
Additive
Effect
NaCl
(Sodium
chloride)
None
PEG
(Polyethylene
glycol)
Wider Size
Selection
Agilent High Sensitivity Chip Trace
1-13
SPRI Based Size Selection
Effects of Common Laboratory Reagents on Size Selection
Table 3 Effects of Common Laboratory Reagents on Size Selection. Blue background represents wider
selection. Red background represents narrower selection.
1-14
Additive
Effect
pH
Narrower Size
Selection for
pH < 3.
No effect for
pH ≥ 3.
Sodium Acetate
None
Agilent High Sensitivity Chip Trace
B24965AA
Glossary
Glossary
B24965AA
bp
base pair
Double Size Selection
a Left and Right Side Size Selection performed on the
same sample
Left Side Size Selection
defines the new start point of the sample’s size
distribution
μg
microgram
μL
microliter
ng/μL
nanograms per microliter
Right Side Size Selection
defines the new end point of the sample’s size
distribution
RT
Room Temperature (15-30°C)
Tris
tris(hydroxymethyl)aminomethane (10 mM, pH 8)
TE
Tris with Ethylenediaminetetraacetic acid
(10 mM Tris, pH 8,1 mM EDTA)
Glossary-1
Glossary
Glossary-2
B24965AA
Index
C
concentration of the additive, 1-10
H
High Sensitivity DNA chip, 1-5
Double Size Selection, 1-8
Left Side Size Selection, 1-3
L
Left and Right Side Size Selection
maximize recovery, 1-2
Left Side Size Selection
maximize recovery, 1-2
M
maximize recovery
Left and Right Side Size Selection, 1-2
Left Side Size Selection, 1-2
Right Side Size Selection, 1-2
R
Right Side Size Selection
maximize recovery, 1-2
S
Sample Prerequisites, 1-2
Sample volume, 1-2
standard buffer solution, 1-2
T
trademarks, i-ii
Index-1
Index
Index-2
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